The recent completion of the genome sequences of a number of bacterial species and several eukaryotes has demonstrated the feasibility and utility of sequencing large genomes. Nucleotide sequencing of the Arapidopsis genome has recently been completed, mapping and sequencing of the rice genome has been nearly completed, and vast quantities of expressed sequence tag information are being obtained from many other plants. This wealth of information provides a powerful tool for application of genetic methods for improving economically important species. However the primary hurdle now is to provide a comprehensive understanding of these sequences and the genetic mechanisms controlling plant growth, development and responses to the environment. The assigning of function to this vast array of sequence information will clearly be the most important and perhaps most time consuming step in plant genomics.
Traditional approaches to assign function to given set of nucleotide sequences such as EST's or various gene/promoter combinations are often not efficient. This is especially true for multi-gene families in which a desired phenotype such as yield, may be determined by only one, or a few of several genes within a gene family. For example in maize the phenotype stalk strength is influenced by the cellulose synthase gene family which can consist of as many as thirty-some sequences in an EST library. Gene knockout methods or transposon tagging are ineffective for multiple gene families and are also time consuming, as it takes approximately four generations and up to three years time before any analysis of function can occur, since rounds of backcrossing and selfing are required to fix a given knockout. Transgene expression for both up and down regulation by transgenics has progressed both in scale and the degree of precision in regulating gene expression. Controlling gene down regulation in transgenic plants has made significant strides with the advent of amplicon, hairpin-loop, and tRNA-like structures which invoke various mechanisms of both transcriptional and Post Transcription Gene Silencing (PTGS) for efficient down regulation. However single gene (vector) transformation using one vector at a time is limited because the analysis of the T0 generation requires follow-up analysis in T1 and subsequent generations. This approach is time consuming when the initial objective is to choose a few candidate sequences for further analysis from among a much larger group of twenty-plus candidate nucleotide sequence combinations. The use of an amplicon-type system in which a virus is used to induce Post Transcription Gene Silencing seems favorable. However, virus induced up and down regulation of expression, particularly for a crop-specific virus system, has only been proven in model species such as Nicotiana benthaminia, and expression characteristics are limited by the host viral genome expression characteristics in a given plant species. Thus there is a need in the art for the ability to test a relatively large number of candidate sequences in a parallel system which relies on fast and efficient insertion of nucleotide sequences into expression cassettes, rapid result return from transformation experiments, medium to high throughput analysis, and efficient use of greenhouse and/or growth chamber space to functionally evaluate nucleotide sequences in plants.
It is an object of the present invention to provide a quick and efficient method of mass scale for analysis of nucleotide function in plants.
It is yet another object of the invention to provide vectors which are designed to effect expression of target DNA sequences in plants including up and down regulation of genes for subsequent analysis of its expression products and resulting phenotypes.
It is yet another object of the invention to provide specific protocols to rapidly and efficiently design and construct appropriate expression cassettes and vectors for appropriate constitutive and/or ectopic or not and/or inducible overexpression or post-transcriptional gene silencing of target native or modified cDNAs sequences.
It is yet another object of the invention to provide for use of multiple vectors in a single transformation protocol to generate multiple transformation events, i.e., a “library of vectors” to scale up analysis.
It is yet another object of the invention to provide for in-planta testing by means of a fast cycling plant line to reduce generation time, and maximize greenhouse space to reduce time to analyze phenotypic traits.
It is yet another object to provide high throughput analysis at a phenotypic, biochemical or molecular level to assign function to nucleotide sequences.